Method for investigating the fate of a test compound or the stateof a biological system by means of nmr of hyperpolarised nmr active nuclei

ABSTRACT

The invention is concerned with Nuclear Magnetic Resonance (NMR) spectroscopy and Magnetic Resonance Imaging (MRI), particularly NMR spectroscopy. It provides hyperpolarisation methods offering enhanced sensitivity of detection over conventional NMR for studying the fate of a test compound in a biological system. The methods are particularly suitable for studying metabolism and toxicity of drugs. The resulting NMR sensitivity increase is advantageous in two key aspects of NMR detection: test compounds can be detected at lower concentrations and substantial time saving can be achieved in cases where extensive averaging is conventionaly employed to increase the signal to noise ratio of the corresponding NMR spectra. The methods can be used for studios that were not practical or not possible using conventional NMR.

[0001] This invention is concerned with Nuclear Magnetic Resonance (NMR)spectroscopy and Magnetic Resonance Imaging, particularly NMRspectroscopy. The spectra of NMR active nuclei vary depending on theirenvironment. The present invention provides a method for obtaininginformation regarding the fate of a test compound, which may beexogenous, e.g. a drug, or an endogenous native compound, in abiological system by enhancing the nuclear polarisation of NMR activenuclei of the test compound (hereinafter termed ‘hyperpolarisation’)prior to NMR or MRI analysis. The invention also provides a method forcarrying out NMR pattern profiling to obtain information on the statusof a biological system.

[0002] The term ‘test compound’ as used hereinafter, refers to acompound that may be exogenous or endogenous to the biological system inwhich it is to be studied and that is of physiological interest, e.g. apotential drug substance, which is not a noble gas nor a so-calledOverhauser MRI (OMRI) contrast agent or other MR Imaging agent. A testcompound must contain at least one NMR active nuclei, i.e. a nuclei withnon-zero nuclear spin.

[0003] The term ‘fate’ as used hereinafter when applied to a testcompound encompasses metabolism, absorption, distribution and excretionin a biological system.

[0004] The term ‘biological system’ as used hereinafter encompasseswhole animals, plants, micro-organisms, isolated organs and tissues,isolated cells or cultured cells isolated sub-cellular organelles andexpressed and/or reconstituted enzyme systems. Samples that may beextracted from biological systems such as whole animals, plants,isolated organs or tissues, include tissue or cell samples, faeces, bodyfluids including but not limited to blood, lymph, urine, semen, breastmilk, cerebro-spinal fluid, sweat, lachrymal or parotid secretions orlavage.

[0005] Due to the genomic revolution, combinatorial chemical librariesand high throughput screening, an exponentially increasing number of newchemical entities is now entering or is already in the trial phasesrequired prior to marketing as new drugs. This rapid evolution ofpotentially beneficial drugs has led to an increased pressure on boththe efficacy and safety evaluation processes. There is an on-goingintensive search for new technologies that may optimise the efficiencyof such evaluations.

[0006] Pharmacokinetic and toxicology testing have two key requirements,which are the identification of metabolites formed from the parentcompound and assessment of the toxicity of both the parent compound andits metabolites. During pre-clinical tests and the clinical trial phasesof drug development, it is essential to investigate (bydetection/monitoring) whether trial drugs themselves or theirmetabolites give rise to adverse reactions in in vitro test systems,animals, healthy volunteers or patients. It is also necessary toascertain whether potentially undesirable and even dangerous reactionsare related to the concentration or distribution of the drug or one ormore of its metabolites in the body. In addition such evaluations may beconducted in selected patients in order to determine whether particulargroups of patients with, for instance, identified defects in one or moredrug metabolising enzymes (which may represent a very small minority ofthe pre-clinical and clinical trial populations) are at an increasedrisk of developing adverse drug reactions. An important aspect of anysuch investigations is to determine the fate of a drug substance once ithas been administered, i.e. its absorption, tissue distribution, rateand site(s) of metabolism, characterisation of structure and relativeabundance of metabolites and routes of excretion. There is a need fornew methods for studying the fate of a test compound.

[0007] One of the methods that can be used to study the fate of a testcompound in a biological system is to identify the structures of itsmetabolites. Current techniques for identifying the structure ofmetabolites rely heavily on mass spectroscopy (MS) in combination withliquid chromatography. However, mass spectroscopy is, on its own, oftennot able to characterise the structure of metabolites fully andunambiguously. Data derived from NMR spectroscopy are oftencomplementary to that obtained from MS and when used in combinationthese techniques may allow the structure of metabolites to bedetermined. Unfortunately, currently NMR is relatively insensitive. Inmany cases, the relatively low sensitivity of NMR creates fundamentalproblems that affect the acquisition time needed in order to achieve adesired signal and the lower limit of detection (LOD) of analyte at adefined signal:noise ratio (e.g. 3:1).

[0008] In practical terms, the poor sensitivity of current NMRtechniques limits their application in absorption, distribution,metabolism and excretion (ADME) studies. During the early stages of drugdevelopment, the supply of candidate drug, and hence its metabolites, islimited and there is often not enough material available for analysis byNMR. In addition, the concentration of metabolites produced by in vitroand in vivo screens is low and often well below the level needed foranalysis by NMR. It is not advisable to increase dosing because theroutes of metabolism may change under such non-physiological conditionsand the metabolites formed will be non-representative of these producedunder standard patient treatment regimes.

[0009] At present, it is necessary to scale up the testing procedure andto concentrate the metabolites from large volumes of biological fluids(e.g. cell culture superntants, organ perfusates, plasma, bile, urineetc.) in order to characterise the new candidate drugs by NMR. This isvery time consuming so, in practice, it often does not take place untillate in the drug development phase. This leads to many of the costlylate phase candidate drug failures. Ideally, NMR needs to beincorporated as a routine analytical tool alongside MS as early aspossible in the evaluation of the ADME characteristics of new chemicalentities. However, this is not feasible with the sensitivity ofconventional NMR techniques. The present invention using hyperpolarisedNMR active nuclei addresses many of the aforementioned limitations andhence offers many advantages compared to conventional NMR techniques, aswill be discussed below.

[0010] In addition to ADME applications, toxicity (Tox) evaluations arealso central to the drug approval process. The current situation withregard to Tox testing is arguably even more problematic than ADME. Manycandidate drugs are found to exhibit unacceptable toxicity late inclinical trials, and even occasionally post launch. It is widelyaccepted within the pharmaceutical industry that current pre-clinicaltoxicological screens are inadequate. Current in vitro screens arepoorly predictive of the in vivo situation. Consequently, the toxicityof new candidate drugs must be evaluated thoroughly in two animalspecies before large scale testing in humans. This is costly and timeconsuming. Moreover, results from animal testing are not alwayspredictive for humans. There is an urgent requirement for improvedtoxicity screening procedures.

[0011] Bioanalytical approaches for evaluating drug efficacy and safetycurrently include measurements of responses of living systems to drugcandidates either at the genetic level or at the level of expression ofcellular proteins, using so-called genomic and proteomic methodsrespectively. However, since both methods ignore the dynamic metabolicstatus of the whole cell, tissue or organism, even in combinationgenomics and proteomics may not provide sufficient information aboutintegrated cellular function in living systems to assess accurately thefate and toxicological profile of a drug candidate. A high-resolution ¹HNMR-based approach has been suggested (Xenobiotica, 1999, vol. 29,1181-1189, J. K. Nicholson et al) and has been termed metabonomics.Metabonomics is defined as the quantitative measurement of the dynamicmultiparametric metabolic response of living systems topathophysiological stimuli or genetic modification. It is anticipatedthat such analyses will highlight patterns of variations of endogenouscompounds produced in response to known toxins. This should enable thetoxicity of new candidate drugs to be predicted by comparison. Themethods according to the present invention using hyperpolarised NMRshould enable improvements in metabonomic analyses of the effect of newcandidate drugs on the distribution, metabolism and excretion ofendogenous compounds in comparison to currently employed techniques.Hyperpolarised NMR will enable the utilisation of ¹³C NMR for thesesorts of studies. Currently, only the use of ¹H NMR is practicable (dueto insensitivity of detection for ¹³C using conventional NMR) and theinformation content of the analysis is limited by the chemical shiftrange of ¹H. In comparison, ¹³C NMR offers a much wider range ofchemical shifts. The improvements obtained by the methods of the presentinvention may be in terms of speed and sensitivity and any combinationthereof.

[0012] NMR pattern profiling is a technique that is used to acquireinformation about the status of a biological system (J Pharm Biomed AnalMarch 1995, 13(3): 205-11, Anthony M L et al; Mol Pharmacol July 1994;46(1): 199-211, Anthony M L et al; Naturwissenschaften January 1975;62(1):10-4, Kowalski B R and Bender C F). Typically an NMR pattern,which may be a spectrum or an image, from a system that has beensubjected to some kind of perturbation in its state is compared with theNMR pattern from the same type of system in its usual state. Changes inthe pattern can then be correlated with the change in state of thesystem. The change in state of the system may be, for example, exposureto a drug substance, change in environment or a disease, or a change instage of development of the system. Profiling can be used to compare twosystems to determine whether they are in the same or in differentstates. Once information is available on the pattern exhibited by a typeof system in a variety of states, profiling can be used to determine thestate of a test system of that type by comparing the pattern exhibitedby the test system with the known patterns for that type of system. Theinformation is conveniently stored electronically and algorithmicanalyses can be used to compare the pattern for the test system with theknown patterns. The algorithmic analyses are suitably carried out usinga computer and appropriate software. NMR pattern profiling ispotentially a powerful technique for acquiring a plethora of informationabout a system even when specific entities, for example metabolites,cannot unambiguously be identified individually. However, its usefulnesshas been limited by the inability of current technology to detect andresolve differences in individual spectra due to the relatively lowsensitivity of the NMR technique. The present invention usinghyperpolarised NMR active nuclei addresses this limitation and therebypotentially enables NMR pattern profiling to be used to obtain moreinformation than is available using conventional NMR techniquesregarding, for example, a system's health, function and metabolicstatus, and the mechanisms occurring within the system.

[0013] The present invention is not limited to any specific method forpolarising NMR active nuclei. Such polarisation may be achieved by manydifferent ways, for example, by polarisation transfer from a noble gas,or by one of the ‘Brute force’ (WO 99/35508, Nycomed Imaging AS), DNP(WO 98/58272, Nycomed Imaging AS) and para hydrogen (p-H₂) methods (WO99/24080, Nycomed Imaging AS) as explained below.

[0014] Noble gas isotopes having non-zero nuclear spin can behyperpolarised, i.e. have their polarisation enhanced over theequilibrium polarisation, e.g. by the use of circularly polarised light.Preferred techniques for hyperpolarisation include spin exchange with anoptically pumped alkali metal vapour and metastability exchange. Noblegases to which this technique can be applied include ³He and ¹²⁹Xe. Theenhanced nuclear polarisation of a noble gas can be transferred toanother NMR active species in close proximity by spin-spin interaction.WO 97/37239 (Lawrence Berkeley National Laboratory) describes methodsfor transferring nuclear polarisation from a hyperpolarised noble gas tonuclear spins on a target compound, leading to an enhancement of thecorresponding NMR or MRI signals. WO 98/30918 (Nycomed Imaging AS)relates to ex-vivo dynamic nuclear polarisation (DNP) or NuclearOverhauser Effect (NOE) cross-polarisation from a hyperpolarised gas toan MRI agent where the gas is separated from the MRI agent prior toadministration to the body.

[0015] Although the NMR spectroscopy or imaging method of the presentinvention provides similar types of information about the fate of a testcompound as conventional NMR or MRI, it offers potential advantages.Among these advantages are: a) increased sensitivity of analysis; and b)increased speed of acquisition of NMR spectra (or images). The analysisof drugs/metabolites or physiological compounds containing an NMR activenuclei may provide additional information previously only supplied bystudying corresponding ¹⁴C-labelled compounds, whilst being free fromthe problems associated with radioactive isotopes.

[0016] Furthermore, in comparison to studies using fluorescent reagentsand related labelling technology, for example, the current inventiondoes not require the synthesis of an adduct comprising a reporter, e.g.a fluor, and the test compound in order to enable detection. Thereforethe present invention offers the following advantages over conventionalfluorescence based detection systems:

[0017] 1. There is no alteration in the chemical structure of noveldrugs or physiological compounds. There is always a disadvantage withtechniques such as fluorescent methods in that the additional chemicalcomponent may influence the measurement. Specifically, the fluorescentlabel may be of a significant size when compared with the test compoundand sometimes as large or larger than the test compound. Consequentlythe fate of the labelled test compound may be quite different from thenon-labelled compound.

[0018] 2. Fluorescence measurement may not be specific, due for exampleto dye leakage, dye compartimentalisation, quenching of signal andautofluorescence.

[0019] Similarly, although the NMR pattern profiling method of thepresent invention provides similar types of information about the stateof a system when compared to existing NMR pattern profiling methods, italso offers potential advantages. Among those possible advantages are:a) increased sensitivity of analysis; and b) increased speed ofacquisition of NMR spectra (or images). Increased detail in patternprofiles, may be realised as a consequence of increased sensitivity suchthat features become visible within spectra that would not bediscernible from noise under conventional NMR. The sensitivity increaseenables utilisation of ¹³C NMR as well as ¹H NMR which collectivelywould provide additional information relative to ¹H NMR profiles alone.Changes in patterns that are not visible in pattern profiles obtainedusing conventional NMR techniques may be observed using the methodaccording to the present invention. These small changes may beparticularly important for example when studying toxicity or carryingout quality assurance testing on cell cultures.

[0020] One aspect of the present invention concerns a method formonitoring any aspect of the fate of a test compound, includingmetabolism, which method comprises polarising one or more NMR activenuclei in the test compound and detecting changes in the spectra of thenuclei. The test compound may be an exogenous compound such as a drug oran endogenous ‘native’ substance. The changes may be detectedcontinuously or as a series of discrete measurements or as a singlemeasurement. Both quantitative and qualitative measurements areincluded, especially the dynamic clearance pattern of any metabolitesusing samples of e.g. exhaled respiratory gases, blood, blood plasma,urine or other body fluids. Suitable nuclei are those with non-zeronuclear spin. Preferred NMR active nuclei are ¹³C, ¹⁵N, ³¹P, ¹⁹F and/or¹H, ¹³C is particularly preferred

[0021] Where the test compound is a drug, and isotopic enrichment isappropriate to facilitate detection it is preferred to use stable,non-radioactive isotopes that have substantially no effect on thetherapeutic efficacy of the drug, such as ¹³C and ¹⁵N. Alternatively,nuclear species occurring at high natural abundance such as ³¹P, ¹⁹Fand/or ¹H, can be detected according to the methods of the invention.

[0022] Where the test compound is exogenous, e.g. a drug, it may bepolarised before administration to the system in which its fate is to bestudied. Alternatively, for endogenous and exogenous test compounds, thewhole system or samples extracted from the system may be subjected to anappropriate hyperpolarisation technique at various times.

[0023] Thus, in a first aspect the present invention provides a methodfor investigating the fate of a test compound containing at least oneNMR active nuclei said method comprising:

[0024] administering the test compound to a biological system;

[0025] hyperpolarising the NMR active nuclei in the system-or in asample extracted from the system; and

[0026] analysing the hyperpolarised system or one or more samplesextracted from the system by NMR spectroscopy and/or NMR imaging.

[0027] NMR spectroscopy is the preferred method of analysis,particularly where samples extracted from the system are to be analysed.

[0028] Suitably, where the system is an animal or perfused organ,samples (e.g. biopsy, necropsy or fluid extract) will be taken and thenhyperpolarised. For example, blood or urine samples may be taken. Thesamples may be purified prior to NMR spectroscopy, but this is notalways necessary. An important advantage of the methods according to thepresent invention is that, unlike with prior art methods, spectroscopycan be carried out directly on the crude biological sample without theneed for fractionation, purification or concentration steps.

[0029] This method is particularly suitable for dynamic studies assamples may be taken at time intervals, hyperpolarised, and then NMRspectra of the various samples can be compared to show changes overtime. Hyperpolarisation may be effected by means of a polarising agentas a single transfer, continuous transfer or intermittent transfer. Anappropriate method for hyperpolarisation will be selected depending onthe nature of the system or sample.

[0030] The test compound is preferably exogenous to the biologicalsystem in which its fate is to be studied, e.g. a drug or drugcandidate.

[0031] In another aspect, the invention provides a method forinvestigating the fate of a test compound containing at least one NMRactive nuclei, which method comprises:

[0032] hyperpolarising the NMR active nuclei in the compound;

[0033] administering the hyperpolarised compound to a biological system;and

[0034] analysing the system or samples extracted from the system by NMRspectroscopy and/or NMR imaging.

[0035] An appropriate method of hyperpolarisation will be selecteddepending on the nature of the test compound.

[0036] NMR spectroscopy is the preferred method of analysis,particularly where samples extracted from the system are to be analysed.The test compound is preferably exogenous to the biological system inwhich its fate is to be studied, e.g. a drug or drug candidate. Again,the samples may be subjected to preliminary steps such as fractionation,purification or concentration prior to spectroscopy, but the fact thatthis is not always necessary is an advantage of this method.

[0037] Suitable NMR active nuclei for use in test compounds for themethods according to the first and second aspects include ¹³C, ¹⁵N, ³¹P,¹⁹F and/or ¹H. ¹³C and ¹⁵N are particularly suitable. ¹³C is the mostpreferred.

[0038] Although it may be possible to employ the methods of theinvention with test compounds containing a natural abundance of the NMRactive nuclei, where the test compound is exogenous, it is preferablyenriched with NMR active nuclei before administration to the system.This may include either selective enrichments of one or more sites, oruniform enrichment of all sites. Enrichment can be achieved by chemicalsynthesis or biological labelling. Preferably, a test compound for usein a method according to the invention is an organic compound comprisingan artificially enriched abundance of, for example, ¹³C, eithergenerally or at least in one specific position, at an abundance of atleast 5%, suitably at least 10%, more suitably at least 50%, preferablyat least 75%, more preferably at least 90% and ideally at approaching100%.

[0039] The present invention also covers the use of test compoundscomprising an artificially-enriched abundance of ¹⁵N of at least 1%,suitably at least 5% more suitably at least 10%, preferably at least 50%and more preferably at least 75% or more, and ideally at approaching100%.

[0040] Enrichment of more than one nuclear species, e.g. ¹³C and ¹⁵N maybe performed in the same test compound.

[0041] Although it might be expected that, because different ¹³C centresin a uniformly enriched test compound relax at very different rates,very different signal intensities would be found in any NMR spectraproduced, surprisingly peaks have been observed even for ¹³C centresthat were expected to relax too rapidly to appear in spectra. Thus, apeak for each carbon centre can be observed in spectra producedaccording to a preferred embodiment of the invention wherein the NMRnuclei are ¹³C and analysis is by spectroscopy.

[0042] The degree of hyperpolarisation of the NMR active nuclei ornuclei according to this invention can be measured by its enhancementfactor compared to thermal equilibrium at spectrometer field andtemperature. Suitably the enhancement factor is at least 10, preferablyit is at least 50 and more preferably it is at least 100. However testmethods where even smaller enhancements are achieved may still beperformed usefully due to the shorter time needed for the totalmeasurement compared with conventional methods. If the enhancement isreproducible and the polarisation/NMR measurement can be repeated, thesignal to noise ratio of an NMR signal can be improved. In such a case,the minimum NMR enhancement factor required depends on: a) thepolarisation technique and b) the concentration of the test compound.The enhancement has to be large enough so that the NMR signal from thetest compound can be detected. In this context, it is clear that anenhancement of 10 or less than 10 that it is achievable in a multi-shotexperiment may be very useful due to the time saved in data acquisitioncompared with conventional NMR.

[0043] The analysis steps of the above mentioned methods may be carriedout by continuous monitoring or as a single discrete measurement or as aseries of discrete measurements that may be carried out at suitableintervals over time. Thus, changes in the spectra or images can bemonitored over time and correlated with dynamic events. Such dynamicevents may include metabolic events, changes in distribution, progressin absorption, and progress in excretion of the test compound Theaforementioned methods may be used to monitor the dynamic fate of anytest compound as well as endogenous metabolites or metabolites ofexogenous test compound using samples from e.g. blood, urine or otherbody fluids.

[0044] When the fate to be studied relates to metabolism, NMRspectroscopy rather than MRI should be used as the method of analysis.It may be possible, by carrying out the analysis over time, to identifymany and preferably all known changes in metabolism or appearance ofindividual metabolites of the test compound. It should be possible toassign specific peaks in the spectrum to known metabolites. Theincreased sensitivity of the technique may result in additional peaks(compared with spectra obtained using conventional NMR) due topreviously unrecognised minor products of metabolism appearing in thespectrum. This is important because even tiny amounts of toxicmetabolites can cause a drug candidate to exhibit damaging side-effects.This method will thus be a very useful tool to evaluate themetabolic/toxicity pattern for a drug or other substances as well asproducing mechanistic information.

[0045] Metabolic studies can be carried out in whole animals, perfusedorgans; tissue or cell cultures or in test tube systems utilising, forexample, microsomal preparations or other sub cellular fractions such asS9 mix (which contains both phase one and phase two metabolisingenzymes). Where whole animals or organs are used, it is preferred toemploy the method according to the first aspect of the invention and tohyperpolarise samples extracted from the animal or organ. Blood andurine samples are particularly suitable. Studying urine samples has theadvantage of enabling cumulative effects to be observed.

[0046] The methods will be particularly useful when some of themetabolites are not previously known. The chemical shift from thepolarised NMR active nuclei may help to identify the nature of the newmetabolites.

[0047] In addition, the methods may also be useful even if it is notpossible to identify the different metabolites unequivocally because insome situations the dynamic clearance pattern from unknown metabolitesmay also have a significant value.

[0048] When the fate of the test compound to be studied is absorption,the system is conveniently a whole animal, perfused organ, tissue orcell system. Whole animals and perfused organs, especially wholeanimals, are particularly suitable. Typically, samples are extractedfrom different locations in the system and analysed by NMR spectroscopy.Alternatively, the whole system can be analysed by NMR imaging. In oneembodiment, the test compound is hyperpolarised and then administered tothe human or animal body by inhalation. Absorption of the test compoundvia the lungs is monitored by NMR imaging. In another embodiment, thetest compound is hyperpolarised, then administered to the human oranimal body by intravenous injection and absorption from the bloodstreamis observed by NMR imaging.

[0049] If it is desired to investigate the distribution of a testcompound, it is preferred to hyperpolarise the compound and thenadminister it to a whole animal or human body. The compound may beadministered by inhalation, or alternatively it can be administeredintravenously. NMR imaging is preferably used to monitor thedistribution of the test compound. Suitably, imaging can be carried outcontinuously. Alternatively, a series of discrete images can be producedover time. NMR imaging can be carried out on the whole human or animalbody. Alternatively, solid sections can be taken from an animal that hasbeen killed at a known time interval from administration of the testcompound and these sections can be imaged. In this case, the solidsections are hyperpolarised before imaging.

[0050] If the fate of the test compound to be investigated is excretion,it is preferred to administer the test compound to a whole animal andthen to extract samples of, for example, bile, saliva, faeces, urine orexhaled air. These samples are hyperpolarised prior to NMR analysis. NMRspectroscopy should be employed in studies of this type.

[0051] In a third aspect, the present invention provides a method forinvestigating the state of a biological system containing at least oneNMR active nuclei, which method comprises:

[0052] hyperpolarising the NMR active nuclei; and

[0053] analysing the system or samples extracted from the system by NMRspectroscopy and/or NMR aging to generate a NMR pattern of the system.

[0054] The hyperpolarisation step may be carried out on the whole systemor on a sample extracted from the system.

[0055] In one preferred embodiment, the following additional steps arecarried out:

[0056] subjecting the system to a change in its state;

[0057] hyperpolarising the NMR nuclei;

[0058] analysing the system or samples extracted from the system in itschanged state by NMR spectroscopy and/or NMR imaging to generate an NMRpattern of the system in its changed state;

[0059] comparing the NMR patterns of the system and the system in itschanged state and identifying any changes in the NMR pattern.

[0060] The changes in NMR patterns identified in the final step can becorrelated with the change in state of the system.

[0061] Alternatively, two or more systems of the same type may bestudied. The first, or test, system is subjected to a change in itsstate, while the second, or control system is not. The hyperpolarisationand analysis steps are carried out on each system and the NMR patternsare compared and any differences between patterns for the test systemand the control system are identified.

[0062] Thus, in the fourth aspect, the present invention provides amethod for investigating the state of a biological system containing atlease one NMR active nuclei which comprises:

[0063] subjecting the system to a change in its state;

[0064] hyperpolarising the NMR nuclei;

[0065] analysing the system or samples extracted from the system by NMRspectroscopy or NMR imaging to generate an NMR pattern of the testsystem;

[0066] comparing the pattern with a pattern obtained from a controlsystem that was not subjected to a change in its state prior tohyperpolarisation and analysis; and

[0067] identifying any differences between the pattern from the testsystem and the pattern from the control system.

[0068] Several test systems can be subjected to the same or differentchanges in state and their NMR patterns can be compared with the patternfrom a single control system.

[0069] The state of the system may be changed by external or internalinfluences. Examples of external influences include exposure to anexogenous substance, such as a drug or other type of test compound, oralterations in the environment of the system, e.g. changes intemperature or pH. An example of an internal influence is thedevelopment of the system over time, e.g. cell growth anddifferentiation.

[0070] Suitably the NMR patterns are stored electronically, for examplein a database. Algorithmic analysis is conveniently used to carry outthe comparison step, typically by employing a computer with appropriatesoftware.

[0071] NMR spectroscopy is the preferred method of analysis. The NMRactive nuclei is suitably a nuclei with non-zero nuclear spin. Preferredactive nuclei are ¹³C, ¹⁵N, ³¹P, ¹⁹F and/or ¹H. ¹³C is particularlypreferred.

[0072] The methods can be repeated to acquire NMR patterns for a systemin a number of different states. This information is conveniently storedelectronically, for example in a database. When one or more NMR patternsare available for a system in a known state or states, these can becompared with the NMR pattern for a system of the same type in anunknown state. If the NMR patterns are substantially similar or the samefor the system in a known state and the system in an unknown state, theunknown state will be the same or similar to the known state. Normally,the more NMR patterns available for a particular type of system in avariety of states, the more likely it is to find a pattern substantiallysimilar or the same as the pattern for a test system of that type in anunknown state.

[0073] Thus, in a fifth aspect, the invention provides a method forinvestigating the state of a test biological system containing at leastone NMR active nuclei, which method comprises:

[0074] hyperpolarising the NMR active nuclei;

[0075] analysing the test system or samples extracted from the testsystem by NMR spectroscopy and/or NMR imaging to generate an NMR patternfor the test system;

[0076] comparing the NMR pattern for the test system with the NMRpattern for at least one other system of the same type as the testsystem, said other system being in a known state when its pattern wasgenerated;

[0077] determining the state of the test system.

[0078] The hyperpolarisation step may be-carried out on the system or ona sample extracted from the system.

[0079] Conveniently, the comparison step is carried out by algorithmicanalysis, for example by using a computer with suitable software.Suitably, the NMR pattern for the test system is compared with severalother NMR patterns and preferably with a significant number of other NMRpatterns stored electronically, for example in a database.

[0080] The accuracy of this method will depend, amongst other factors,on the similarity of the test system with the systems of the same typefor which NMR patterns are available. Thus, more accurate results willbe obtained regarding the state of isolated test cells where the cellsare derived from the same lineage, tissue and species as the cellcultures for which patterns are known, than if the test cells arederived from a different cell lineage, tissue or species. The method isparticularly useful for quality assurance testing of systems that areintended to be the same, e.g. cell cultures, wherein small butphysiologically significant differences can be detected.

[0081] The methods of the third, fourth and fifth aspects are useful forinvestigating responses of a biological system to a compound, e.g. adrug, about which relatively little is known. Useful data about theeffects of the compound can be obtained by comparing the NMR pattern ofa system exposed to it in various quantities with the NMR patterns forthe system when exposed to other compounds. For example, the patternsobserved for cells derived from human liver when exposed to a testcompound can be compared with NMR patterns for the same cells whenexposed to substances with known effects on the liver. If the patternfor cells exposed to the test compound is similar to the pattern forcells exposed to a compound that is known to have liver toxicity, it islikely that the test compound will also exhibitliver toxicity. This isparticularly useful when a baseline of pre and post testing strategiescan be established using the same system. NMR profiling can also provideimportant structural information about unknown compounds.

[0082] The profiling methods of the third, fourth and fifth aspects areparticularly suitable for studying plants.

[0083] A polarised noble gas, preferably ³He or ¹²⁹Xe, or a mixture ofsuch gases, may be used according to the present invention to effectnuclear polarisation of the test compound or system comprising at leastone NMR active nuclei. The hyperpolarisation may also be achieved byusing an artificially enriched hyperpolarised noble gas, preferably ³Heor ¹²⁹Xe. The hyperpolarised gas may be in the gas phase, it may bedissolved in a liquid, or the liquefied hyperpolarised gas itself mayserve as a solvent. Alternatively, the gas may be condensed onto acooled solid surface and used in this form, or allowed to sublime.Either of these methods may allow the necessary intimate mixing of thepolarised gas with the target to occur. In some cases, liposomes ormicrobubbles may encapsulate the hyperpolarised noble gas.

[0084] In a further embodiment, the present invention provides a methodwherein the polarisation may be imparted to atoms of significance in thetest compound or system (e.g. ¹³C, ¹⁵N, ³¹P, ²⁹Si, ¹⁹F and ¹H isotopes)by thermodynamic equilibration at a very low temperature and high field.Hyperpolarisation compared to the operating field and temperature of theNMR spectrometer is effected by use of a very high field and very lowtemperature (Brute force). The magnetic field strength used should be ashigh as possible, suitably higher than 1T, preferably higher than 5T,more preferably 15T or more and especially preferably 20T or more. Thetemperature should be very low e.g. 4.2K or less, preferably 1.5K orless, more preferably 1.0K or less, especially preferably 100 mK orless. It will be appreciated that this embodiment is not suitable forpolarising a viable biological system where that system is a wholeanimal, isolated organ or tissue or cultured cells.

[0085] In a further embodiment, the present invention provides a methodfor polarisation transfer using the DNP method effected by a DNP agent,to effect nuclear polarisation of the test compound or system comprisingat least one NMR active nuclei. DNP mechanisms include the Overhausereffect, the so-called solid effect and the thermal mixing effect.

[0086] Most known paramagnetic compounds may be used as a “DNP agent” inthis embodiment of the invention, e.g. transition metals such aschromium (V) ions, magnesium (II) ions, organic free radicals such asnitroxide radicals and trityl radicals (WO 98/58272) or other particleshaving associated free electrons. Where the DNP agent is a paramagneticfree radical, the radical may be conveniently prepared in situ from astable radical precursor by a radical-generating step shortly before thepolarisation, or alternatively by the use of ionising radiation. Duringthe DNP process, energy, normally in the form of microwave radiation, isprovided, which will initially excite the paramagnetic species. Upondecay to the ground state, there is a transfer of polarisation to a NMRactive nuclei of the target material. The method may utilise a moderateor high magnetic field and very low temperature, e.g. by carrying outthe DNP process in liquid helium and a magnetic field of about 1T orabove. Alternatively, a moderate magnetic field and any temperature atwhich sufficient NMR enhancement is achieved in order to enable thedesired studies to be carried out may be employed. The method may becarried out by using a first magnet for providing the polarisingmagnetic field and a second magnet for providing the primary field forMR spectroscopy/imaging. It will be appreciated that, as in the Bruteforce method described above, that this embodiment is not suitable forpolarising a viable biological system where that system is a wholeanimal, isolated organ, or tissue or cultured cells if high fields andlow temperatures are used.

[0087] It might be expected that the presence of a paramagnetic radicalwould cause line-broadening and susceptibility shifts in NMR spectraproduced in analysing the sample. Pleasingly, this does not occur in theexperiments carried out to date. This good result may be explained bythe low relaxivity of the radical used and its low concentration in thefinal sample. Therefore it is preferred to use a DNP radical with lowrelaxitivity in those embodiments of the invention where a DNP radicalis required.

[0088] In a further embodiment, the present invention provides a parahydrogen induced polarisation method. Hydrogen molecules exist in twodifferent forms, para hydrogen (p-H₂) where the nuclear spins are antiparallel and out of phase (singlet state) and ortho hydrogen (0-H₂)where the spins are parallel or anti parallel and in phase (tripletstate). At room temperature, the two forms exist in equilibrium with a1:3 ratio of para:ortho hydrogen. However, preparation of para hydrogenenriched hydrogen can be carried out a low temperature, 160K or less, inthe presence of a catalyst. The para hydrogen formed may be stored forlong periods, preferably at low temperature, e.g. 18-20K. Alternativelyit may be stored in pressurised gas form in containers which have aninner surface which is non-magnetic and non-paramagnetic.

[0089] The preparation of a para hydrogen-containing species is achievedby exposing an unsaturated precursor (containing NMR active nuclei) ofthe compound to the para hydrogen-enriched hydrogen gas in the presenceof a suitable catalyst. This enriched hydrogen will then react with theprecursor by reduction imparting a non-thermodynamic spin configurationto the target molecule. The compounds suitable for use are thus preparedfrom precursors which can be reduced by hydrogenation and which willtherefore typically possess one or more unsaturated bonds, e.g. doubleor triple carbon-carbon bonds.

[0090] When the p-H₂ molecule is transferred to the precursors of thecompound (by means of catalytic hydrogenation with e.g. (PPh₂)₃RhCl),the proton spins remain anti parallel and begin to relax to thermalequilibrium with the normal constant T1 of the hydrogen in the compound.However, during relaxation some of the polarisation may be transferredto neighbouring nuclei by pulse sequence Progress in NuclearSpectroscopy, 31, (1997), 293-315), low field cycling or other types ofcoupling. The presence of the NMR active nuclei as e.g. ¹³C (and ¹⁵Netc) with a suitable substitution pattern close to the relaxing hydrogenmay lead to the polarisation being trapped in the slowly relaxing ¹³C(and ¹⁵N etc) resulting in a high enhancement factor.

[0091] This embodiment is suitable for the aspects of the invention inwhich the NMR active nuclei in the test compound is hyperpolarised priorto administration to the system in which its fate is to be tested.

[0092] A further hyperpolarisation transfer embodiment of this inventionis the spin refrigeration method. This method covers spin polarisationof a solid compound or system by spin refrigeration polarisation. Thesystem is doped with or intimately mixed with a suitable paramagneticmaterial such as Ni²⁺, lanthanide and actinide ions in crystal form witha symmetry axis of order three or more. The instrumentation is simplerthan that required for DNP, with no need for a uniform magnetic fieldsince no resonant excitation field is applied. The process is carriedout by physically rotating the sample around an axis perpendicular tothe direction of the magnetic field. The pre-requisite for this methodis that the paramagnetic species has a highly anisotropic g-factor. As aresult of the sample rotation, the electron paramagnetic resonance willbe brought in contact with the nuclear spins, leading to a decrease inthe nuclear spin temperature. Sample rotation is carried out until thenuclear spin polarisation has reached a new equilibrium. Again, it willbe appreciated that this embodiment is not suitable for polarising abiological system where that system is a whole animal, isolated organ ortissue or cultured cells.

[0093] When a test compound, system or sample from a system has beenhyperpolarised, it is desirable to preserve as much as possible of thepolarisation prior to NMR analysis. Some of the hyperpolarisationtechniques described above are only effective when transferringpolarisation in the solid state. However, it is often desired toinvestigate the NMR spectrum of a compound sample or system in theliquid state, in order to improve spectral resolution and sensitivity.Alternatively, line-narrowing techniques such as Magic Angle Spinning(MAS) can be employed to increase spectral resolution of NMR in thesolid state and enable low temperature NMR analysis.

[0094] If the compound, sample or system is not solid, it mayconveniently be frozen in an appropriate solvent mixture prior topolarisation transfer by one of the methods that needs to be carried outin the solid state. Solvent mixtures have been found to be particularlysuitable, especially if the mix forms an amorphous glass. The amorphousmatrix is employed to ensure homogenous intimate mixing of radical andtarget in the solid while the sample is subject to DNP polarisation.

[0095] If a liquid state NMR technique is to be employed, once thecompound, sample or system has been hyperpolarised, it can be rapidlyremoved from the polarisation chamber and then dissolved in a suitablesolvent. It is advantageous to use solvents that will not interfere withthe images or, more usually, the spectra produced in the analysis step.Deuterated solvents such as D₂O are particularly suitable. Stirring,bubbling, sonification or other known techniques can be used to improvethe speed of dissolution. Suitably, the temperature and pH of thesolution are maintained to allow optimal dissolution and a long nuclearrelaxation time.

[0096] Preferably, the compound, sample or system and then the solutionthereof are kept in a holding field throughout the period betweenpolalrisation and analysis in order to prevent relaxation. A holdingfield provides a field higher than the Earth's magnetic field andsuitably higher than 10 mT. It is suitably uniform in the region of thesample. Although a holding field is not required for all test compounds,much better results are obtained for some test compounds when such afield is used and it is difficult to predict in advance which compoundswill require such a holding field, especially if the structure of thecompound is not known a priori. Therefore, it is preferable to use aholding field whenever a system or sample is polarised and thentransferred for analysis. The optimal conditions will depend on thenature of the compound, sample or system. The solution is subsequentlytransferred for examination by standard solution phase NMR analysis. Thetransfer process is preferably automated. Alternatively, thepolarisation transfer and dissolution steps are suitably integrated intoa single automated unit. In an additional suitable embodiment, thepolarisation transfer and sample dissolution steps are automated and NMRdetection hardware is also housed within the same single fullyintegrated unit. A holding field will not be required with such a fullyintegrated system.

[0097] Different ¹³C centres relax at very different rates.Consequently, very different signal intensities would be expected toappear in the resulting NMR spectra if nuclear relaxation occurs duringthe transfer from the hyperpolarisation unit to the NMR spectrometer.Surprisingly, peak heights from different centres within uniformly ¹³Cenriched molecules have been observed to be of the same order. Apossible explanation is that the effect may be due to a redistributionof the enhanced polarisation by cross-relaxation at certain carboncentres. This is useful, because it allows more information to beobtained than may otherwise have been expected. It is not unreasonableto assume that any carbon centre within a given test compound will bedetected with similar sensitivity.

[0098] Alternatively, where a solid state NMR technique is to be used,the solid state compound, sample or system may be hyperpolarised, e.g.by DNP, brute force, spin refrigeration transfer or any other methodthat will work in the solid state at low temperature. Subsequently, thehyperpolarised sample will be moved into a solid-state MAS NMR probe.The movement is suitably rapid and is conveniently carried out vialifting or ejection. The sample in the NMR probe will then be spun sothat high-resolution solid state NMR spectroscopy can be carried out.The entire process can be automated and will preferably be carried outin an integrated unit.

[0099] The invention will now be illustrated by reference to thefollowing non-limiting examples.

EXAMPLES

[0100] Trityl Radical

[0101] Stable triarylmethyl reagents are particularly suitable for DNPenhancements. The radical used in the following examples is tris(8-carboxyl-2,2,6,6,-tetra(2(1-hydroxyethyl))benzo[1,2-d:4,5-d′]bis(1,3)dithiol-4-yl)methylsodium salt (hereafter called trityl radical). This was made accordingto the methods described in WO98/39277.

[0102] Stock solutions of trityl radical were prepared in deuteratedglycerol for each example.

[0103] To glycerol-D₈ (200 μl) was added trityl radical (6.28 mg). Theradical was dissolved by stirring under gentle heating and briefsonication and was stored in a closed vial until required. This yieldeda stock at 22 mM which was used in the study of benzoic acid (example1); the final trityl radical concentration used for the DNP step was13.2 mM. Samples of trityl radical solution were gently warmed tofacilitate subsequent dispensing. Similarly a stock of trityl radicalwas prepared at 22.25 mM and this was employed for the hippuric acid inurine sample (example 4) with a final radical concentration of 14.9 mMfor the DNP step. Additionally a stock was prepared at 25 mM and thiswas used for the remaining studies (examples 2,3,5 and 6), with finalradical concentrations of 15 mM for the DNP steps.

[0104] 40% ^(W)/v Sodium deutoxide was purchased from SIGMA/ALDRICHchemical company and was diluted 100 fold in D₂O to prepare a 0.4%^(W)/v. stock which was used in examples 1,2 and 3.

[0105] Glycerol-D₈, D₂O and DMSO-D₆ reagents were purchased fromSIGMA/ALDRICH

Example 1

[0106] Study of (¹³C-carboxyl) Benzoic Acid Isolated From Rat Urine.

[0107] (¹³C-carboxyl) Benzoic acid was purchased from SIGMA/Aldrichchemical company. A sample was added to rat urine at approximately 5 mgper ml and then isolated by solid phase extraction (SPE) and reversedphase high performance liquid chromatography (RP hplc, C18 Kromasil,25×1 cm, 5 μm) using gradient elution with formic acid in water andformic acid in methanol. The isolated material was then dried and analiquot re-analysed by RP hplc indicating a purity of 94%, with the maincomponent co-eluting with carrier material, monitored by on-line UVdetection (at 254 nm) and yielding the expected (M—H)⁻ ion at 122 massunits by on-line electrospray ionisation mass spectrometry (EI-MS).

[0108] A sample of (¹³C-carboxyl) benzoic acid (89 μg, 0.72 μmol)obtained as described above was dissolved in sodium deutoxide in D₂O(0.4% ^(W)/v, 7.5 ul, approximately 1.5 equivalents) and D₂O (7.5 μl).Trityl radical dissolved in glycerol-D₈ (24 μl, 22 mM) was added to thebenzoic acid solution and the subsequent cocktail was mixed tohomogeneity with a disposable plastic pipette. The sample was rapidlyfrozen, as small droplets, by dripping via a fine plastic pipette into aliquid nitrogen bath. The frozen sample drops were collected using smalltweezers and placed in a liquid nitrogen cooled Kel-F cup and this wastransferred for sample polarisation. The sample was polarised overnightat a magnetic field of 3.354T and at a microwave frequency of 93.925GHz. Microwave power was 100 mW and sample temperature was maintained at1.25K for the duration of polarisation. The test sample was dissolved inhot D₂O (approximately 5 ml) in situ and a portion (approximately 1 mlin a 5 mm NMR tube, estimated sample temperature is 333K) was rapidlytransferred to an INOVA 400 MHz spectrometer for measurement of a liquidstate NMR spectrum. The sample was exposed to the earth's magnetic fieldduring transit to the spectrometer (approximately 20 seconds transfertime). The signal to noise estimated for the carboxyl carbon of(¹³C-carboxyl) benzoic acid peak, observed at 176.0 ppm is approximately440. The NMR spectrum was obtained in a single acquisition (acquisitiontime was 1.2 seconds, sweep width 25 kHz, following a RF pulse of 6microseconds, with WALTZ proton de-coupling applied during the pulse andacquisition; line broadening of 1 Hz was applied and signal to noise wasdetermined by Varian Vnmr software). The same sample was subsequentlyanalysed by non enhanced NMR with proton decoupling in the samespectrometer. A thermal equilibrium signal for the carbonyl carbon wasobtained with a signal to noise of approximately 8. This controlspectrum was acquired in 37 hours (averaging 168000 scans with a scanrepetition rate of 0.8 seconds and a flip angle of 11.2 degrees i.e.under Ernst angle conditions.

[0109] The sample used for NMR analysis was retained and subsequentlyre-analysed by RP hplc (C18 Kromasil, 25×0.46 cm, 5 μm) with gradientelution with formic acid in water and formic acid in acetonitrile and byEI-MS).

[0110] Re-analysis indicated predominantly (¹³C) benzoic acid and thematerial yielded the expected molecular ion by MS analysis.

[0111] Results

[0112] The spectrum of FIG. 1 was obtained. The prominent peak from thelabelled ¹³COOH site of benzoic acid is clearly visible at 176.0 ppm.The prominent signals at around 71.9 ppm and around 62.3 ppm areidentified as solvent peaks from glycerol. The acquisition time for thisspectrum was a matter of seconds, compared to the days that would berequired by conventional NMR. The spectrum illustrates an exceptionalsignal to noise ratio.

[0113] Conclusions

[0114] The observed signal to noise ratio of the enhanced NMR spectrum,compared with the signal to noise ratio of the conventional NMR spectrumfrom the same sample, confirms that the method according to theinvention gives substantial improvement. The enhancement is estimated tobe of the order of a few thousandfold. Similarly, the remarkably shortdata acquisition time is evidence that the methods of the invention maybe used to carry out studies that would simply be too time-consuming todo in practice using conventional NMR.

[0115] The lines of the spectrum are very narrow and are positioned asexpected in the spectrum. Therefore, it can be inferred that the DNPradical is not affecting the quality of the NMR spectrum. This isimportant because it shows that the signal enhancement obtained by usingthis hyperpolarisation method of the invention is not compromised byartefacts in the enhanced spectrum obtained.

Example 2

[0116] Study of (¹³C-carboxyl) Hippuric Acid (Primary Benzoic AcidMetabolite) Isolated from Rat Urine After iv Administration of(¹³C-carboxyl) Benzoic Acid.

[0117] Embodiment Without a Holding Field Magnet

[0118] (¹³C-carboxyl) Benzoic acid was administered intravenously at 10mg/Kg (at 0 and 2 hrs) to 4 anaesthetised rats; urine was collected fromcanulated urethra. The major metabolite (¹³C-carboxy) hippuric acid wasisolated by SPE and RP-hplc. The isolated material was then dried and analiquot re-analysed by RP hplc indicating a purity of 99%, with the maincomponent co-eluting with authentic carrier material, monitored byon-line UV detection (at 254 nm) and yielding the expected (M−H)⁻ion at179 mass units by on-line electrospray ionisation mass spectrometry(EI-MS).

[0119] A sample of (¹³C-carboxyl) hippuric acid (463 μg, 2.57 μmol)obtained as described above was dissolved in sodium deutoxide in D₂O(0.4% ^(W)/v, 17.5 μl, approximately 0.97 equivalents). Trityl radicaldissolved in glycerol-D₈ (26 μl, 25 mM) was added to the hippuric acidsolution and the subsequent cocktail was mixed to homogeneity with adisposable plastic pipette. The sample was rapidly frozen, as smalldroplets, by dripping via a fine plastic pipette into a liquid nitrogenbath. The frozen sample drops were collected using small tweezers andplaced in a liquid nitrogen cooled Kel-F cup and this was transferredfor sample polarisation. The sample was polarised for 4 hours at amagnetic field of 3.354T and at a microwave frequency of 93.925 GHz.Microwave power was 100 mW and sample temperature was maintained at1.25K for the duration of polarisation. The test sample was dissolved inhot D₂O (2-3 ml) in situ and a portion (approximately 1 ml in a 5 mm NMRtube, estimated sample temperature is 333K) was rapidly transferred toan INOVA 400 MHz spectrometer for measurement of a liquid state NMRspectrum.

[0120] Results

[0121] The NMR spectrum was obtained in a single acquisition(acquisition time was 1.2 seconds, sweep width 25 kHz, following a RFpulse of 6 microseconds; ¹H decoupling was employed as above; linebroadening of 1 Hz was applied and signal to noise was determined byVarian Vnmr software). A very noisy NMR spectrum from Hippuric acid wasobtained. The signal to noise estimated for the carboxyl carbon peak of(¹³C-carboxyl) hippuric acid, observed at approximately 170 ppm is lessthan 4. Strong glycerol signals were observed.

[0122] Results

Example 3

[0123] Study of (¹³C-carboxyl) Hippuric Acid (Benzoic Acid Metabolite)Isolated from Rat Urine After iv Administration of (¹³C-carboxyl BenzoicAcid.

[0124] Embodiment Utilising a Holding Magnet

[0125] A sample of (¹³C-carboxyl) hippuric acid (463 μg, 2.57 μmol)obtained as described above was dissolved sodium deutoxide in D₂O (0.4%^(W)/v, 17.5 μl, approximately 0.97 equivalents). Trityl radicaldissolved in glycerol D₈ (26 μl, 25 mM) was added to the hippuric acidsolution and the subsequent cocktail was mixed to homogeneity with adisposable plastic pipette. The sample was rapidly frozen, as smalldroplets, by dripping via a fine plastic pipette into a liquid nitrogenbath. The frozen sample drops were collected using small tweezers andplaced in a liquid nitrogen cooled Kel-F cup and this was transferredfor sample polarisation. The sample was polarised for 4 hours at amagnetic field of 3.354T and at a microwave frequency of 93.925 GHz.Microwave power was 100 mW and sample temperature was maintained at1.25K for the duration of polarisation. The test sample was dissolved inhot D₂O (2-3 ml) in situ and a portion (approximately 1 ml in a 5 mm NMRtube, estimated sample temperature is 333K) was rapidly transferred toan INOVA 400 MHz spectrometer for measurement of a liquid state ¹³C NMRspectrum. The sample was maintained in a magnetic holding field of 10 mTduring transit to the spectrometer (approximately 20 seconds transfertime). The signal to noise estimated for the carboxyl carbon of(¹³C-carboxyl) hippuric acid peak, observed at 171.2 ppm, isapproximately 1500. An NMR spectrum was obtained in a single acquisition(acquisition time was 1.2 seconds, sweep width 25 kHz, following a RFpulse of 6 microseconds; ¹H decoupling was employed as above linebroadening of 1 Hz was applied and signal to noise was determined byVarian Vnmr software).

[0126] The sample used for NMR analysis was retained and subsequentlyre-analysed by RP hplc (C18 Kromasil, 25×0.46 cm, 5 μm) with gradientelution with formic acid in water and formic acid in acetonitrile and byEI-MS). Re-analysis indicated predominantly (¹³C) hippuric acid and thematerial yielded the expected molecular ion by MS analysis.

[0127] Results

[0128] The enhanced NMR spectrum is shown in FIG. 2. A much highersignal to noise ratio for the hippuric acid NMR signal was obtained inthis example using a holding field than in Example 2 where no holdingfield was used.

[0129] Pleasingly, peaks in addition to that expected for the label wereobserved.

[0130] The chemical shift difference of the NMR signal from the hippuricacid label compared to that from the benzoic acid label was as expectedfrom conventional NMR studies.

[0131] Conclusions

[0132] It is beneficial to use a holding field when analysing somecompounds by a technique where the sample is polarised in one locationand NMR analysis takes place in another. Since it is not always possibleto predict in advance which compounds will benefit from the use of sucha holding field, it should be used routinely when ex situhyperpolarisation techniques are employed.

[0133] The presence of additional peaks from the hippuric acid in theenhanced NMR spectrum indicates that the methods of the invention aresuitable for studying test compounds at lower doses than would bepossible using the conventional NMR. This is potentially significant forstudying toxicity and or metabolism of drugs and drug candidates, wheresome metabolites may be present only at very low concentrations.

[0134] Moreover, it can be inferred from the presence of additionalpeaks that the methods of the invention may be suitable for use withtest compounds wherein there is a lower degree of enrichment or evennatural abundance of the NMR active nuclei.

[0135] The observation of NMR signals at the expected chemical shiftpositions confirms that this method of the invention does not introduceartefacts and can be used to study the fate of the test compound.

Example 4

[0136] (¹³C-carboxyl) Hippuric Acid (Analysed Directly After Spikinginto Rat Urine)

[0137] (¹³C-carboxyl) Hippuric acid was synthesised in-house from(¹³C-carboxyl) benzoic acid and glycine.

[0138] A sample was added to rat urine at approximately 5 mg/ml, whichwas the level measured in urine (0-2 hr collection) in the benzoic acidmetabolism study described above. A sample containing 95 μg of synthetic(¹³C-carboxyl) hippuric acid was dissolved in approximately 20 μl of raturine. Trityl radical dissolved in glycerol-D₈ (30 ul, 22.25 mM) wasadded to the hippuric acid solution and the subsequent cocktail wasmixed to homogeneity with a disposable plastic pipette. The sample wasrapidly frozen, as small droplets, by dripping via a fine plasticpipette into a liquid nitrogen bath. The frozen sample drops werecollected using small tweezers and placed in a liquid nitrogen cooledKel-F cup and this was transferred for sample polarisation. The samplewas polarised for 4 hours at a magnetic field of 3.354T and at amicrowave frequency of 93.925 GHz. Microwave power was 100 mW and sampletemperature was maintained at 1.25K for the duration of polarisation.The test sample was dissolved in hot D₂O (2-3 ml) in situ and a portion(approximately 1 ml in a 5 mm NMR tube, estimated sample temperature is333K) was rapidly transferred to an INOVA 400 MHz spectrometer formeasurement of a liquid state ¹³C NMR spectrum. The sample wasmaintained in a magnetic holding field of 10 mT during transit to thespectrometer (approximately 20 seconds transfer time).

[0139] The signal to noise estimated for the carboxyl carbon of(¹³C-carboxyl) hippuric acid peak, observed at 171.1 ppm, isapproximately 534. An NMR spectrum was obtained in a single acquisition(acquisition time was 1.2 seconds, sweep width 25 kHz, following a RFpulse of 6 microseconds ¹H decoupling was employed as above; linebroadening of 1 Hz was applied and signal to noise was determined byVaran Vnmr software).

[0140] The sample used for NMR analysis was retained and subsequentlyre-analysed by RP hplc (C18 Kromasil, 25×0.46 cm, 5 um) with gradientelution with formic acid in water and formic acid in acetonitrile and byEI-MS). Re-analysis indicated predominantly (¹³C) hippuric acid and thematerial yielded the expected molecular ion by MS analysis.

[0141] Results

[0142] Two NMR signals were observed, one arising from hippuric acid at170.7 ppm and one smaller singlet NMR signal at 163.1 ppm that can beassigned tentatively to urea, because its chemical shift is consistentwith the shift predicted using ACD Labs software, and this substance isknown to be a major constituent of urine.

[0143] Conclusions

[0144] In this case, a test compound was analysed directly in abiological matrix, i.e. rat urine. The fact that a clear signal wasobtained is very encouraging and confirms that samples collected overtime could be analysed, thus enabling dynamic studies. Anotherconclusion is that samples may not need to be fractionated prior to NMRanalysis.

[0145] It is not unreasonable to infer that had benzoic acid also beenpresent in the sample, it would also have been detected. Accordingly,pharmacokinetic studies could be undertaken using the methods of theinvention.

Example 5

[0146] (U-¹³C)Paracetamol Isolated from Rat Urine

[0147] (U-¹³C)Paracetamol was synthesised. This material was added torat urine at approximately 5 mg per ml and then isolated by solid phaseextraction (SPE) and reversed phase high performance liquidchromatography (RP hplc, C18 Kromasil, 25×1 cm, 5 μm) using gradientelution with formic acid in water and formic acid in methanol.

[0148] The isolated material was then dried and an aliquot reanalysed byRP hplc indicating a purity of 94%, with the main component co-elutingwith carrier material monitored by on-line UV detection (at 254 nm) andyielding the expected (M+H)⁺ ion at 160 mass units by on-lineelectrospray ionisation mass spectrometry (EI-MS).

[0149] A sample of (U-¹³C) paracetamol (312 μg, 1.96 μmol) obtained asdescribed above was dissolved in DMSO-D₆ (10 μl) and D₂O (14 μl). Tritylradical dissolved in glycerol-D₈ (36 μl, 25 mM) was added to the (U-¹³C)paracetamol solution and the subsequent cocktail was mixed tohomogeneity with a disposable plastic pipette. The sample was rapidlyfrozen, as small droplets, by dripping via a fine plastic pipette into aliquid nitrogen bath. The frozen sample drops were collected using smalltweezers and placed in a liquid nitrogen cooled Kel-F cup and this wastransferred for sample polarisation. The sample was polarised for 4hours at a magnetic field of 3.354T and at a microwave frequency of93.925 GHz. Microwave power was 100 mW and sample temperature wasmaintained at 1.25K for the duration of polarisation. The test samplewas dissolved in hot D₂O (2-3 ml) in situ and a portion (approximately 1ml in a 5 mm NMR tube, estimated sample temperature is 333K) was rapidlytransferred to an INOVA 400 MHz spectrometer for measurement of a liquidstate ¹³C NMR spectrum. The sample was maintained in a magnetic holdingfield of 10 mT during transit to the spectrometer (approximately 20seconds transfer time). An NMR spectrum was acquired in a singleacquisition (acquisition time was 1.2 seconds, ¹H discoupling wasemployed as above, sweep width 25 kHz, following a RF pulse of 6microseconds).

[0150] The sample used for NMR analysis was retained and subsequentlyre-analysed by RP hplc (C18 Kromasil, 25×0.46 cm, 5 um) with gradientelution with formic acid in water and formic acid in acetonitrile and byEI-MS). Re-analysis indicated predominantly (¹³C) paracetamol and thematerial yielded the expected molecular ion by MS analysis.

[0151] Results

[0152] An enhancement was observed with all peaks being present atpositions consistent with prediction.

Example 6

[0153] Study of (U-¹³C)paracetamol-sulphate Isolated from Rat Bile Afteriv Administration of (U-¹³C)paracetamol

[0154] (U-¹³C)Paracetamol was administered intravenously at 20 mg/kg (at0 and 3 hrs) to 4 anaesthetised rats, urine was collected from canulatedurethra and bile was collected from canulated bile duct (2 rats only) at0-3 and 3-6 hours. Bile (3-6 hrs) was extracted with dichloromethane andthen fractionated by RP-hplc (C18 Kromasil, 25×1 cm, 5 um) usinggradient elution with formic acid in water and formic acid in methanol).

[0155] Several metabolites were collected. Paracetamol sulphate wasidentified by on-line EI-MS, from its (M+H)⁺ ion at 240 mass units. Theisolated material was then dried and an aliquot re-analysed by RP hplcindicating a peak purity of 96.7%, monitored by on-line UV detection (at254 nm) and gave the expected (M+H)+ ion at 240 mass units by on-lineelectrospray ionisation mass spectrometry (EI-MS).

[0156] A sample of (U-¹³C) paracetamol-sulphate (100 g, 0.42 μmol)obtained as described above was dissolved in D₂O (24 μl). Trityl radicaldissolved in glycerol-D₈ (36 μl, 25 mM) was added to the (U-¹³C)paracetamol solution and the subsequent cocktail was mixed tohomogeneity with a disposable plastic pipette. The sample was rapidlyfrozen, as small droplets, by dripping via a fine plastic pipette into aliquid nitrogen bath. The frozen sample drops were collected using smalltweezers and placed in a liquid nitrogen cooled Kel-F cup and this wastransferred for sample polarisation. The sample was polarised for 4hours at a magnetic field of 3.354T and at a microwave frequency of93.925 GHz. Microwave power was 100 mW and sample temperature wasmaintained at 1.25K for the duration of polarisation. The test samplewas dissolved in hot D₂O (2-3 ml) in situ and a portion (approximately 1ml in a 5 mm NMR tube, estimated sample temperature is 333K) was rapidlytransferred to an INOVA 400 MHz spectrometer for measurement of a liquidstate ¹³C NMR spectrum. The sample was maintained in a magnetic holdingfield of 10 mT during transit to the spectrometer (approximately 20seconds transfer time). An NMR spectrum was acquired in a singleacquisition (acquisition time was 1.2 seconds, sweep width 25 kHz,following a RF pulse of 6 microseconds ¹H decoupling was applied asabove).

[0157] The sample used for NMR was retained and subsequently re-analysedby RP hplc (C18 Kromasil, 25×0.46 cm, 5 μm) with gradient elution withformic acid in water and formic acid in acetonitrile and by EI-MS).Re-analysis indicated a major component with a retention time consistentwith paracetamol sulphate from previous analysis and the materialyielded the expected molecular ion by MS analysis.

[0158] Results

[0159] The enhanced NMR spectrum is shown in FIG. 3. Differences wereobserved in the spectrum for paracetamol sulphate compared withparacetamol. In particular, the chemical shift positions for ¹³C NMRsignals arising from aromatic sites are significantly different.

[0160] The peak heights for the carbon centres were of the same order.

[0161] Conclusions

[0162] The paracetamol sulphate had been produced by metabolism in a ratand was collected from bile. Bile is a different biological matrix thanurine (from which the hippuric acid and benzoic acid were collected. Theresults indicate that NMR enhancements can be observed irrespective ofthe biological matrix from which the test compound is derived. The factthat a different spectrum was obtained compared with paracetamolconfirms that it may be possible to differentiate between peaks fromparent compounds and their metabolites in a mixture, even in the case ofsubtle structural differences between them.

[0163] A pleasing observation was that, as in all the other examples, nosignals arising from the radical, were observed.

[0164] It was surprising to see that the peak heights for the carboncentres were of the same order. Since different carbon centres decay atvery different rates, it was expected that the signal intensities forthe carbon centres would be very different. Indeed, it was not expectedto be possible to observe the methyl peaks at all. Therefore, moreinformation was obtained from the experiment than anticipated. It is notunreasonable to assume that any carbon centre within a given testcompound would be detected with similar sensitivity by the method of theinvention.

1. A method for investigating the fate of a test compound containing atleast one NMR active nuclei which method comprises: hyperpolarising theNMR active nuclei; administering the test compound to a biologicalsystem in which its fate is to be studied; and analysing the system or asample extracted from the system by NMR spectroscopy or NMR imaging. 2.A method for investigating the fate of a test compound containing atleast one NMR active nuclei which method comprises: administering thetest compound to a biological system in which its fate is to be studied;hyperpolarising the NMR active nuclei in the system or in a sampleextracted from the system; and analysing the hyperpolarised system orsample by NMR spectroscopy and/or NMR imaging.
 3. A method according toclaim 1 or claim 2 wherein the test compound is enriched with NMR activenuclei.
 4. A method according to any of claims 1 to 3 wherein the NMRactive nuclei are selected from ¹³C, ¹⁵N, ³¹P, ¹H and ¹⁹F.
 5. A methodaccording to any of claims 2 to 4 wherein the hyperpolarised system orsample is retained in a holding field in the period fromhyperpolarisation to analysis.
 6. A method according to any of claims 1to 5 wherein the NMR nuclei are ¹³C and the analysis is carried out byNMR spectroscopy, characterised in that a peak is observable for eachcarbon centre in the test compound.
 7. A method according to any ofclaims 1 to 6 wherein the metabolism of a test compound is studied.
 8. Amethod according to claim 5 wherein the system is a whole animal orhuman body, and wherein samples, e.g. blood or urine, are extracted fromthe system over time, each of said samples being hyperpolarised and thenanalysed by NMR spectroscopy.
 9. A method according to any of claims 1and 3 to 6 for studying the absorption of a test compound wherein thesystem is a whole animal or human body and the system is analysed by NMRimaging over time.
 10. A method according to any of claims 2 to 6 forstudying absorption of a test compound wherein the system is a wholeanimal or human body and wherein samples, e.g. blood or urine, areextracted from the system over time, each of said samples beinghyperpolarised and then analysed by NMR spectroscopy.
 11. A methodaccording to any of claims 1 and 3 to 6 for studying the distribution ofa test compound wherein the system is a whole animal or human body, thehyperpolarised test compound is administered by inhalation orintravenous injection, and the distribution of the test compound in thebody is analysed by NMR imaging.
 12. A method according to any of claims2 to 6 for studying the excretion of a test compound wherein the systemis a whole animal or human body and samples, e.g. urine samples areextracted over time, each of said samples being hyperpolarised and thenanalysed by NMR imaging.
 13. A method for investigating the state of abiological system containing at least one NMR active nuclei, whichmethod comprises: hyperpolarising the NMR active nuclei in the system;and new line analysing the system or samples extracted from the systemby NMR spectroscopy or NMR imaging to generate an NMR pattern for thesystem.
 14. A method according to claim 13 wherein the method furthercomprises: subjecting the system to a change in its state;hyperpolarising the NMR active nuclei; analysing the system or samplesextracted from the system in its changed state by NMR spectroscopyand/or NMR imaging to generate an NMR pattern for the system in itschanged state; comparing the NMR patterns for the system and the systemin its changed state and identifying any changes in the NMR pattern. 15.A method for investigating the state of a biological system containingat least one NMR active nuclei which method comprises: subjecting thesystem to a change in its state; hyperpolarising the NMR nuclei;analysing the system or samples extracted from the system by NMRspectroscopy or NMR imaging to generate an NMR pattern from the system;comparing the pattern with a pattern obtained from a control system thatwas not subjected to a change in its state prior to hyperpolarisationand analysis; and identifying any differences between the pattern fromthe test system and the pattern from the control system.
 16. The methodaccording to claim 14 or 15 wherein the test system is subjected to achange in its state by exposure to a drug.
 17. A method forinvestigating the state of a biological system containing at least oneNMR active nuclei which method comprises: hyperpolarising the NMR activenuclei in the test system; analysing the test system or samplesextracted from the test system by NMR spectroscopy or NMR imaging togenerate an NMR pattern for the test system; comparing the NMR patternfor the test system with at least one other system of the same type asthe test system, said other system being in a known state; anddetermining the state of the test system